Early detection of illness is essential in preventing symptoms from escalating and infectious diseases from spreading. Electrochemical biosensors are a promis- ing tool in healthcare detection. Previously, the collaboration between the Arold and Inal labs has led to the design of organic electrochemical transistors (OECT) capable of rapidly detecting coronavirus in saliva by using nanobody constructs as biorecognition units. In this project, I aimed to prove the versatility of nanobody- functionalized OECT biosensors in detecting other relevant viruses, specifically, Zika and Dengue. Both viruses pose a risk to multiple populations around the world, including the Kingdom of Saudi Arabia. I designed and produced nanobod- ies that are reported to bind to the NS1 glycoprotein, which is released by Zika and Dengue into the blood of the patient. Then, I confirmed the binding of the nanobodies to their associated targets. I also developed a robotic liquid handling script to automate the biosensing operations. Ultimately, this project aims to support the design of a multiplex OECT biosensor for blood-borne pathogens.

Genetic regulatory circuits control metabolism, development, and environmental response across all kingdoms of life. Genetic circuit engineering facilitates sustainable and efficient production of biopharmaceutical, chemical, fiber, and food products that keep humans healthy, nourished, and clothed. However, the complexity of most genetic regulatory circuits, particularly in the context of multicellular eukaryotes, often prevents them from being leveraged as tools or applied technologies with bioeconomic relevance. However, synthetic biology enables the transfer of genes, circuits, networks, and even whole chromosomes between organisms. This approach can be leveraged to port genetic circuits into simple model organisms to control existing and engineer new cellular functions. Still, porting genes to non-native contexts can affect circuit function due to unknown factors. For this reason, iterative design-build-test-learn (DBTL) cycles are necessary for optimizing circuits in new contexts. To facilitate the DBTL cycle, automation approaches can be deployed for streamlining synthetic genetic circuits optimization. Here, I provide a case study for how using synthetic biology and automation – a biofoundry approach – has facilitated engineering of the auxin signaling pathway in a synthetic yeast system. Auxin is a phytohormone involved in nearly every aspect of plant growth and development, and this striking versatility designates it as a target for biotechnology development and a candidate for engineering. First, I provide a literature review of the history of synthetic auxin engineering in yeast, a survey of tools available for expanding yeast synthetic biology, and a summary of applicable automation tools and platforms. Next, I describe and validate a platform called AssemblyTron, which deploys liquid handling robotics for DNA assembly and can serve as the foundation of a biofoundry platform. I then introduce TidyTron, which is a protocol library for automated wash and reuse of single use lab plastics to promote biofoundry sustainability. Next, I expand the AssemblyTron package by providing protocols for mutant and modular indexed plasmid library assembly. Finally, I describe a modular indexed plasmid library (toolkit) for rapid assembly of auxin circuit variants and validate it by building and optimizing an auxin circuit. / Doctor of Philosophy / Genetic mechanisms allow humans, plants, and microbes to grow, breathe, speak, and survive. The DNA that encodes these genetic mechanisms produces protein machines that make chemicals, transfer them, and respond to them in other cells. This process is called signaling, and the protein machines involved make a circuit. In biotechnology, we harness natural genetic circuits to create important products like biopharmaceuticals, food, and clothes. However, the genetic circuits that make valuable proteins/chemicals are usually located on chromosomes along with every other gene involved in building an advanced, multicellular organism (called the genome). Synthetic biology allows us to choose just the DNA that encodes a genetic circuit of interest and put it into the chromosome of a simpler organism with faster growth, smaller genome, etc., which allow us to engineer it more easily. However, transferring a gene circuit to a new organism can cause problems, and it is usually necessary to try many versions of gene circuits to find one that works. Using robots to do synthetic biology can make it faster and less error-prone, which enables more versions of the genetic circuit to be tested. Here, I describe a biofoundry approach where I combined synthetic biology and robotics to speed up the process of building and optimizing the auxin plant hormone signaling pathway. Auxin is a small molecule that plants produce and transfer throughout their leaves, stems, and roots to turn growth on or off (e.g., auxin causes plants to do things like bend towards the sun). I focus on auxin because my goal is to manipulate the auxin pathway to rationally control plant growth. First, I provide a recap of existing work in the field of auxin synthetic biology, tools for transferring auxin circuits into simpler organisms, and available robotics that can speed up auxin synthetic biology. Next, I introduce a software called AssemblyTron, which I developed for building and modifying genes (a process called DNA assembly) with a robot. Next, I discuss how I used the same robot to wash and reuse plastic pipette tips and plates to improve lab sustainability. I then discuss an extended version of AssemblyTron that can be used for more advanced DNA assembly applications like making 10s – 100000s of versions of gene circuits at the same time. Finally, I introduce a collection of auxin circuit DNA parts that can be assembled interchangeably for rapid synthetic auxin engineering.

The extent of devastation caused by a biological warfare attack is highly correlated to the time from release to detection. As a step towards lowering the detection time the international project TWOBIAS was launched. Here, the main goal is to develop an automated, specific and sensitive combined detection and identification instrument capable of identifying a biological threat within an hour. The identification unit is comprised of a sample preparation module, an amplification module and a detection module and utilizes a proximity ligation assay in combination with circle-to-circle amplification in order to detect a biological threat. This thesis describes the automation of the sample preparation steps of the assay and the integration with the downstream units. The functionality of the sample preparation module was verified by subjecting it to biological samples in a laboratory and at a real-life location. The results showed that the sample preparation module was capable of preparing a sample collected in a complex environment with the same results as a sample prepared in a laboratory.

Ziel dieser Arbeit war die Erhöhung der Temperaturtoleranz des Cyanobakteriums Synechocystis sp. PCC 6803 mittels ungerichteter Mutagenese und adaptiver Evolution. Trotz des erneuten Interesses an Cyanobakterien und Mikroalgen in den letzten Jahren, gibt es nur relativ wenige aktuelle Studien zum Einsatz dieser Methoden an Cyanobakterien. Zur Analyse eines mittels Mutagenese erzeugten Gemischs an Stämmen, ist es von großem Vorteil Hochdurchsatz-Methoden zur Kultivierung und zum Screening einsetzen zu können. Auf Basis eines Pipettierroboters wurde solch eine Plattform für phototrophe Mikroorganismen neu entwickelt und folgend stetig verbessert. Die Kultivierung erfolgt in 2,2ml Deepwell-Mikrotiterplatten innerhalb einer speziell angefertigten Kultivierungskammer. Schüttelbedingungen, Beleuchtung, Temperatur und CO2-Atmosphäre sind hierbei vollständig einstellbar.Die Plattform erlaubt semi-kontinuierliche Kultivierungen mit automatisierten Verdünnungen von hunderten Kulturen gleichzeitig. Automatisierte Messungen des Wachstums, des Absorptionsspektrums, der Chlorophyllkonzentration, MALDI-TOF-MS sowie eines neu entwickelten Vitalitätsassays wurden etabliert. Für die Mutagenese wurden die Letalität- und die nicht-letale Punktmutationsrate von ultravioletter Strahlung und Methylmethansulfonat für Synechocystis charakterisiert. Synechocystis wurde mit den so ermittelten optimalen Dosen mehrfach behandelt und anschließend einer in vivo Selektion unterzogen. Somit wurde dessen Temperaturtoleranz um bis zu 3°C erhöht. Über die Screeningplattform wurden die thermotolerantesten monoklonalen Stämme identifiziert. Nach einer Validierung wurde das vollständige Genom der Stämme sequenziert. Hierdurch wurden erstmals Mutationen in verschiedenen Genen mit der Langzeittemperaturtoleranz von Synechocystis in Verbindung gebracht. Bei einigen dieser Gene ist es sehr unwahrscheinlich, dass sie mittels anderer Verfahren hätten identifiziert werden können. / The goal of this work was the increase of the thermal tolerance of the cyanobacteria Synechocystis sp. PCC 6803 via random mutagenesis and adaptive evolution. Even with the renewed interest in cyanobacteria in the recent years, there is relatively limited current research available on the application of these methods on cyanobacteria. To analyse a mixture of various strains typically obtained through random mutagenesis, a method allowing high-throughput miniaturized cultivation and screening is of great advantage. Based on a pipetting robot a novel high-throughput screening system suitable for phototrophic microorganisms was developed and then constantly improved. The cultivation was performed in 2,2 ml deepwell microtiter plates within a cultivation chamber outfitted with programmable shaking conditions, variable illumination, variable temperature, and an adjustable CO2 atmosphere. The platform allows semi-continuous cultivation of hundreds of cultures in parallel. Automated measurements of growth, full absorption spectrum, chlorophyll concentration, MALDI-TOF-MS, as well as a novel vitality measurement protocol, have been established. Prior to the mutagenesis, the lethality and rate of non-lethal point mutations of ultraviolet radiation and methyl-methanesulphonate were characterized for Synechocystis. The thus determined optimal dosages were applied to Synechocystis followed by in vivo selection in four rounds of mutagenesis, thereby raising its temperature tolerance by 3°C. The screening platform was used to identify the most thermotolerant monoclonal strains. After validation, their whole genomes were sequenced. Thus mutations in various genes were identified which promote the strains'' thermal tolerance. For some of the genes it is very unlikely that their link to high thermal tolerance could have been identified by other approaches.